Cyclodextrins are cyclic molecules consisting of 1-4 linked alpha-D-glucopyranose monomeric units. The cyclodextrins containing 6-, 7-, and 8-membered rings, commonly known as alpha-, beta-, and gamma-cyclodextrin, respectively, are the most important cyclodextrins to date, possibly because of their availability relative to cyclodextrins of different ring size. The usefulness of these cyclodextrins arises from their ability to reversibly form inclusion complexes, or clathrates, with many types of compounds. Inclusion complexes arise when a host molecule, such as cyclodextrin, has a structure containing an interior cavity into which guest molecules can bind by weak interactions such as van der Waal's forces. The latter are short range forces which are sufficiently strong to allow the formation of definite, generally solid complexes, but are sufficiently weak to permit ready dissociation of the complex to a host and guest molecule.
The cyclodextrins are doughnut-shaped molecules with an interior cavity whose size and shape is determined by the number of glucose units that make up the ring. In alpha-cyclodextrin the almost cylindrical cavity is approximately 7 angstroms deep and 5 angstroms in diameter. In beta-cyclodextrin the depth is the same but the diameter is 7 angstroms, and in gamma-cyclodextrin it is again 7 angstroms deep but 9 angstroms in diameter. Cyclodextrins are soluble in water because of the many hydroxyl groups of the glucose subunits that surround the rim of the cavity. However, the interior of the cavities themselves are hydrophobic, and these hydrophobic cavities extract organic molecules from aqueous solution if the organic materials have the correct shape and hydrophobic character.
The complexing ability of cyclodextrins lends itself to various uses. For example, the cyclodextrins are used in encapsulating desirable flavors and fragrances which can then be stored for reasonably long periods of time and added to foods at their preparation. Reciprocally, cyclodextrins may be used in removing undesirable flavors and fragrances from food by complexing with them. Cyclodextrins also are used in the protection of foods against oxidation, photochemical degradation, and thermal decomposition. These and other uses have been summarized by J. Szejtli, Starch, 34, 379-385 (1982)
To date cyclodextrins have been prepared by treating starch with a cyclodextrin glycosyltransferase (CG) first at a high temperature to liquefy the starch, then at a lower temperature to form the cyclodextrins from the liquefied starch. Although many variations are possible all utilize a liquid starch of low dextrose equivalent (DE), less than about 4, as a substrate for the enzyme. The prior art methods have been described and summarized by K. Horikoshi, Process Biochemistry, May, 1979, 26-30, and by M. Matzuzawa et al., Die Starke, 27, 410-413 (1975).
For continuous production of cyclodextrins as well as for minimizing enzyme cost and maximizing enzyme utilization a process using a cyclodextrin glycosyltransferase immobilized as a fixed or fluidized bed would be advantageous. In such a process the use of liquefied starch as a feed is an undesirable limitation, because the low starch solubility, on the order of 1% w/v, limits both cyclodextrin productivity (the amount of cyclodextrin formed per unit time) and cyclodextrin concentration in the product mixture. A high cyclodextrin concentration in the product mixture is desirable to facilitate subsequent cyclodextrin purification. Although a suspension of liquefied potato starch has been used as a feedstock for soluble CG, it is an unacceptable feedstock for a bed of immobilized cyclodextrin glycosyltransferase (IMCG) which would effectively behave, as a filtering aid to remove the suspended particles, ultimately leading to bed plugging.
With the above in mind it was thought that use of thinned starch, that is, a partially hydrolyzed starch, as the feedstock for IMCG might be beneficial. Initial experiments quickly demonstrated different limitations characteristic of this new feed. Thus, cyclodextrin conversion decreases with increasing dextrose equivalent and increasing dry solids content. This results from enzyme inhibition by glucose initially present in the partially hydrolyzed starch and which also is formed by various disproportionation reactions effected by CG itself. It was also observed that cyclodextrin formation passes through a maximum which arises from a slow hydrolysis of cyclodextrin catalyzed by CG, a reaction which produces glucose further inhibiting cyclodextrin formation by the enzyme.
Our results suggested that a process using partially hydrolyzed starch as a feed for IMCG required maximizing the dry solids to maximize the cyclodextrin concentration in the product mix and cyclodextrin productivity, but at the same time minimizing glucose formation, or at least minimizing the effects of glucose inhibition on CG activity. Because the conversion of partially hydrolyzed starch to cyclodextrins is substantially lower than that with liquefied starch, any process utilizing partially hydrolyzed starch as the feedstock for IMCG needs to provide a means of reusing the oligosaccharides in the product mixture as a feed for IMCG so as to provide for the efficient and economical utilization of thinned starch as a feedstock, but without any detrimental effects on cyclodextrin formation.
Our solution to the two aforementioned problems leads to processes for the efficient, economical, and continuous production of cyclodextrins from thinned starch using immobilized cyclodextrin glycosyltransferase. In particular, the process of our invention separates glucose and/or cyclodextrins from the effluent of an IMCG reactor and recycles the oligosaccharide-rich stream to the IMCG reactor. Where only cyclodextrins are separated they are formed under conditions where glucose formation, and consequent enzyme inhibition, is minimized.
One advantage of the processes which are our invention is the efficient use of enzymes. Because the CG is effectively reused, the enzyme cost is substantially reduced. Another advantage is to afford a method of producing cyclodextrins in a continuous process. Still another advantage is that the relative proportion of the components in the mixture of cyclodextrins that is formed may be controlled somewhat by changing the reaction conditions. Yet another advantage is that the processes described herein afford good quality control over the cyclodextrins produced. Our processes afford high purity beta-cyclodextrin in good yield from a readily available and relatively inexpensive feedstock, all of which are highly advantageous.